med microbio--MODULE 13. AEROBIC GRAM POSITIVE RODS (GPR)

Describe the morphology and arrangement of C. diphtheriae when Gram stained.

Morpho: Gram pos rods, pleomorphic

Arrangement: Palisade (picket fence) appearance, chinese letter configuration

State the name of the disease caused by Corynebacterium diphtheriae.

• which strains cause disease and what is main cause of the infection symps

Toxin producing strains (not all strains) cause diphtheria

• Toxin that organism makes is the problem, not organism itself

Relate the pathogenicity of C. diphtheriae to the production of an exotoxin.

• if tox is not present, what does that mean for the strain

• what body site is targeted for damage

exotoxin that blocks protein synthesis in euk cells

• If tox is not present, strain is not pathogenic

• Causes damage to heart and its muscle action

source (3) and transmission diphtheria 

• opp or obligate?

• patho species

• only natural jost

• another name 

Source: soil, water, on skin and mucosal membranes (mouth) in humans and animals as normal flora/ nonpatho

• Called diphtheroids

• Found in skin, respiratory tract (upper), mouth

• Can cause opportunistic infections

• Pathogenic species= C. diphtheria

• Humans are only natural host

Transmission: inhalation

Describe what is meant by “pseudomembrane” in the relation to infection with C. diphtheriae.

• Psudomembrane forms in throat (is tenacious and strong–not easily punctured–so can lead to suffocation and closing of airway in active, advanced cases)

clinical infect of diphtheria: incubation, initial symps, bac colonize where, tox name that effects CNS and its effects on that system, keystone symp

Clinical infection:

• Incubates 2-7 days

• Initial symps are sore throat, fever, malaise (a lot of prez are sim so important for diff diag)

• Bac colonize mucosal memb

• Cytotoxic exotoxin is prod, which enters blood and causes damage to heart and CNS

• Psudomembrane forms in throat (is tenacious and strong–not easily punctured–so can lead to suffocation and closing of airway in active, advanced cases)

2 complications of diphtheria and mortality 

• Myocarditis, suffocation

• 10-30% mortality

State the way by which outbreaks of diphtheria have been prevented over the past years.

• how long is immunity

• double treatment of what

Immunity and prev:

• After recovery, life long immunity

• Major mech by which outbreaks have been prev is by DPT (diphtheria, pertussis, tetanus) series of immunizations

  • Diphtheria component of DPT series is diphtheria toxoid

  • Effectiveness of D in DPT series is very good

Treatment: antibi and antitox

Describe laboratory diagnostics for diphtheria including stains and toxin assessment.

• grows on what culture media (general and one that is selective/diff for C. diphtheriae)

• presence of what allows for definitive ID of patho C. diphtheriae

• toxin ID test: what does a pos result look like

Lab diag:

• Grows on ordinary culture media like BAP

• Specific media avail

  • Tox prod is optimal at Fe conc of .14 microg/mL but is inhib at .5 microg/mL

  • Important since only tox prod strains are patho

  • Loeffler’s media, Tinsdale agar

  • Cystine-tellurite BAP

    • Selective and diff for C. diphtheriae

    • Inhib normal throat flora, and allows for growth of C. diphtheriae

  • Biochemical ID: 

    • Exotox present is definitive ID of patho C. diphtheriae

    • Toxin is ID by ELEK test

• ELEK: in vitro toxigenicity test

  • In vitro diphtheria toxin detection procedure

  • Based upon principle of immunodiffusion (diffusion comp is same as Ab sus testing)

  • Performance of ELEK test:

    • Heavy inoculum of test organism (ctrls and unknowns) is streaked onto plated medium in a single line

    • Antitoxin impregnated in a strip of sterile filter paper and placed on the surface of the inoculated agar medium at right angle to line of inoculum streak

    • Incubate for 24 hrs

    • Arches of ID indicate Ab-Ag reaction and thus toxin production

      • Due to structure being formed bc so much Ag-Ab rxn

      • Antitox is Ab to toxin Ag

distinguishing morpho feature of C. diphtheriae

• stain most commonly perf in lab

Distinguishing feature: metachromatic granules viable with methylene blue stain or granule stain (less common in lab) or even with low rez, gram stains

Given that a patient has C. diphtheriae in his/her throat, identify the definitive test that determines whether the patient has the disease, diphtheria. (order of 2 tests)

First do cystine-tellurite BAP to see if tox prod (black colonies is toxin prod at Fe conc), then do ELEK to conf in conjunction with metachromatic granules viable with methylene blue stain (done first)

Describe the morphology of Bacillus anthracis when Gram stained.

• spores present in vivo?

• looks sim to what other bac—what is diff

Morpho: aerobic gram pos rod, spores are formed when organism is present in the env, no spores are present in vivo (when in wound or inhal)

• Looks sim to clostridium with other clinical sim, but aerobic

• Used by Koch to dev postulates

State the source, 2 species of med importance and transmission of B. anthracis in human infection.

• 3 forms associated w/ transmission type and gen symps of each

Source: commonly found in soil

Medically important species: B. antracis, B. cereus 

Transmission: 

1. Direct contact: cutaneous form

   1. Occurs in indiv with wounds such as skin cuts, abrasions, insect bites that become infected with anthrax spores

      1. Aerosolized spores can be carried by wind

   2. Dark areas appear in the center of a wound, which ulcerates and dries, forming a black nodular area known as “eschar” or “black eschar”-->very characteristic

   3. Usually infection remains localized and complications do not occur, but can have lasting scars

   4. Huge inflammation reaction of infection seen on skin with redness and warmth and swelling

2. Inhal: pulm form

   1. AKA woolsorter’s disease bc of occupational association

      1. Inhal of spores contacted  by handling animal fibers and hides that contain spores–why patient history is important

      2. Resp infections w fever and cough

      3. Rapid progression to resp distress and cyanosis (blueing of skin bc low O2)

      4. Toxins enter blood stream

      5. Toxemia, death

3. Ingestion: GI form

   1. Occurs when spores are ingested

   2. Symps are abdom pain, nausea, anorexia, vomiting (similar to many other disease states)

   3. Bc this form of disease is more diff to diag, the fatality rate is higher than cutaneous form

   4. Toxins may enter blood

   5. Toxemia, and death may follow–but patients usually tend to survive

Describe the major symptoms associated with “woolsorter’s disease” (pulmonary infection with B. anthracis).

Rapid progression to resp distress and cyanosis (blueing of skin bc low O2)

ca of disease, lab diag, culture results anthrax

• appearance on BAP, hemolysis, margin shape, colony characteristics

B. anthracis in general:

disease= anthrax

Lab diag: DNA hybridization with tech that became avail in 2001 (primary pop used to only be sheep wool farmers and soil tillers)

Culture results: aerobic, nonhemolytic, “ground glass” (not shiny) colonies on BAP that are tenacious (sticky: hold form when lifted) and have an undulated margin that show curling (“medusa head” in low power microscope) described as comma-shaped protrusions

Diff between anthrax and nonanthrax bac with similar appearance:

in terms of motility, capsules, length of chains, colony char, heme

anthrax= nonmotile, capsulated, grow in long chains, medusa head colony, no hemolysis 

3 exotox and causes what symps(how many can be made) and synergistic effects or no? of B. anthracis

• Numerous exotoxins with synergistic effects:

  • Edema factor (EF): causes edema, hemorrhage

  • Protective antigen (PA): involved in edema prod

  • Lethal factor (LF): responsible for resp center depression, dec resp, death

• Up to 50 diff tox prod so important to organism’s survival bc invest E

treatment (3), immun against what and how long, prev of anthrax (3—last one has 2 vacc , one for human and one for an)

Treatment: penicillin, doxycycline, fluroquinolines (“cipro”) 

Immunity: lifelong against exotoxins

Prev:

• Isolation of patients w disease (quarantine)

• Specimen handling precautions

• Vacc:

  • Animal–avirulent (live organism but no longer patho) strain of B. anthracis

  • Human–toxoid (antitox sim to a vacc) from exotoxin complex and annual boosters

2001 anthrax attack importance

• BSL

• methods of clean up

• how many spores are infectious 

Anthrax attack:

• Realized we were unprepared

• Spores can survive long time in env after release so clean up was time consuming and costly

  • Chemical methods (ox and nonox–destroy spores)

  • BSL 4 equipment needed

  • Had to clean all spores bc small doses are infectious

  • Can spread via air so wild areas were harder to clean up

Describe the cutaneous manifestations associated with an external infection with B. anthracis.

• characteristic symo

• does it remain loc or sys?

• complications?

Direct contact: cutaneous form

1. Occurs in indiv with wounds such as skin cuts, abrasions, insect bites that become infected with anthrax spores

   1. Aerosolized spores can be carried by wind

2. Dark areas appear in the center of a wound, which ulcerates and dries, forming a black nodular area known as “eschar” or “black eschar”-->very characteristic

3. Usually infection remains localized and complications do not occur, but can have lasting scars

4. Huge inflammation reaction of infection seen on skin with redness and warmth and swelling

Define eschar and relate it to a disease state

Dark areas appear in the center of a wound in cutaneous anthrax caused by B. anthracis, which ulcerates and dries, forming a black nodular area known as “eschar” or “black eschar”-->very characteristic but generally localized and no comp follow

Identify the three disease states caused by B. anthracis.

Cutaneous, pulm, GI anthrax all caused by how they were transmitted–corresponding to location

Bacillus cereus Describe the clinical infection of this bacterium.

• most common prez and 4 other infectious states

• 2 forms

• Localized infections of the eyes, burn sites, traumatic wounds

• Bacteremia, septicemia

• Pneumo

• Meningitis

• Food poisoning (most common prez):

  • Emetic form (vom)

  • Diarrheal form

    • Clinical prez of gastroenteritis

diarrheal form vs emetic form of B. cereus

• which is heat stable tox

• sources

• disease lasts how long

• which has longer incubation (>6 hrs)

• Emetic form: vomiting

  • Due to heat stable emetic toxin (re-heating cooked sources does not remove or denature toxin

  • Source is fried or boiled rice stored at room temp

  • Incubates <6 hrs

  • nausea, vomiting

  • Lasts 8-10 hrs

• Diarrheal form:

  • Due to heat labile enterotoxin (can heat to get rid of tox)

  • Source is meats, soups, veggies, sauces

  • Incubates >6 hrs

  • Abdom pain, diarrhea

  • Lasts 20-36 hrs

State the source of B. cereus. Discuss causative agents of B. cereus disease.

Cooked rice stored at room temp (emetic form) or meats, soups, veggies, sauces (diarrheal form)

Depends on the form: emetic is rice, diarrheal is meats/soups/veggies/sauces

Describe the treatment and prevention of B. cereus-associated infections.

• infections (2 antibi rez) vs food poisoning

• opp or obligate?

• Infections: opportunistic infect

  • Antibi–typically rez to penicillin and cephalosporins

• Food poisoning: 

  • Typically treatment not needed since infections are self-limiting

State the major genus from which this bacterium (Listeria monocytogenes) needs to be discerned and state why there is concern about these two genera of bacteria.

• what 3 things can diff, what 3 things make sim

Looks like strep bc beta heme and lancet shape, and same risk pop (beta strep target neonates and most common cause of meningitis–all sim to listeria)

• Differentiate w high salt conc, umbrella motility at room temp, growth at 4 deg C

Discuss the laboratory diagnostics of Listeria monocytogenes.

• morpho: gram stain, shape

• hemolysis

• temp

• salt conc

• motility pattern

• which are unique to organism

Lab diag:

• Small gram pos rod (almost looks like cocci), lancet shaped that resembles strep

• Growth with w beta-hemolysis

• Capable of growth and 4 deg C

• Grows at high salt conc

• Exhibits umbrella pattern of motility at room temp (23-25 deg C) but not at human body temp (35-37 deg C)

  • Jello agar so can see bac swim out

• * last three bullet points diff from strep bc they don’t do

Risk pop Listeria monocytogenes (3)

• early vs late onset in youngest group

• 2 effects on female group

• Pregnant women (premature labor, septic abortion)

• Newborn infants:

  • Early onset= sepsis

  • Late onset=meningitis

• Elderly and immunocomp (more general to any disease)

Discuss key characteristics of Listeria monocytogenes. (3)

• Capable of growth and 4 deg C

• Grows at high salt conc

• Exhibits umbrella pattern of motility at room temp (23-25 deg C) but not at human body temp (35-37 deg C)

  • Jello agar so can see bac swim out

Listeriosis: symps, CNS involvement, transmission type to infants from mother

• Flu-like symptoms occur, such as fever and chills

• CNS involvement: infection can spread to nervous system and cause headache, stiff neck, confusion, loss of balance, convulsions

•  vertical transmission of listeriosis from pregnant mother to infant by crossing of the placental barrier

  •  fetal listeriosis can lead to miscarriage, stillbirth, systemic infections, granulomatosis infantisepticum

  • Incidence of listeriosis among pregnant women is 13 times higher than the general population

  • This foodborne illness can cause significant fetal and perinatal complications including miscarriage, preterm labor, stillbirth, as well as neonatal listeriosis and possible neonatal death

  •  recalls of listeria contaminated food products have highlighted the potential for pregnant women to be exposed to the bacteria 

Fetal listeriosis: early onset (other name) vs late onset

• which one has better survival

• characteristic symp of early onset

• birth weight if dev in utero

• 2 diseases that follows late onset

• Early onset disease (granulomatosis infantiseptica)

  •  infected infant presents with serious illness at birth, potentially fatal

  •  premature delivery is common baby is born with low birth weight

  •  maybe tends to have sepsis, respiratory distress, fever, rash– a disseminated rash with small, pale, granulomatous nodules is characteristic 

• Late onset disease

  • Encountered bacteria during birth through the birth canal

  •  neonates usually full term and were Healthy Babies In Utero

  •  infant becomes infected late in utero or during delivery: symptoms appear hours to days after birth

  •  meningitis or sepsis follows

  •  chance of survival for late onset is better than for early onset disease 

Describe the source, transmission, treatment and prevention of Listeria-associated infections.

• passive carriers and location of harboring passively in body

• 2 transmissions

• 3 antibi therapies

• prev: neonates and food infect

Source:

• Animals carry this bac in intestine and do not become ill

• Eating contam food can lead to human infect

  • Listeria grows at fridge temp (4 deg C) and tol high salt conc (most ready to eat foods have)

  • Is leading cause of fatal food borne infect in US

• Outbreaks of listeriosis are assoc w/ ready-to-eat foods such as hot dogs, lunch meats, cold cuts, sausage, deli style meat and poultry (all freq served cold and unheated)

• Bac is destroyed if reheated to steaming hot before ingestion

  • Do not drink unpasteurized milk or soft cheeses that are frequently made from pasteurized milk, preheat ready to eat food to steaming hot

Transmission:

•  animals carry listeria monocytogenes and their intestines without becoming sick, their meat is then used and processed→ eaten unheated

•  vertical transmission of listeria occurs from pregnant women to fetus (vertical= in utero, during birth, colostrum, suckled milk transmission)

  •  horizontal transfer is when transferred for one person to another 

•  eating foods contaminated with listeria can lead to human infection

Treatment:

• Antibiotics such as ampicillin and aminoglycoside initially used as a combo strategy

•  Penicillins

•  Macrolides

Prev:

•  for neonates: treat mother with antibiotics during pregnancy

  •  detected and pre-delivery check up two to three weeks to delivery date– same as Group B strep

• For food infections:

  •  wash hands and surfaces with hot soapy water, listeria grows in refrigerator so always clean up refrigerator spills with hot sudsy water

  •  do not cross contaminate

    •  separate utensils for ready to eat foods, raw meats, fresh vegetables and fruit

  •  reheat ready to eat meats and poultry until steaming hot

  •  refrigerate and freeze perishables within 2 hours, including ready to eat foods 

what are diptheroids?

normal flora corynebacterium on skin and resp tract that are opportunistic pathos

which bac secretes exotox tha blocks prot synth in euk cells

C. diphtheriae

which bac forms a psudomemb

C. diphtheriae

which bac resembles strep but is aerobic and with other lab diffs

L. monocytogenes

late or early onset of fetal listeriosis has a better chance of survival?

late